Does nitric oxide derived from spemn function during the Ca^<2+>-transient at fertilization of sea urchin eggs?

海胆卵受精时胞内Ca^2+浓度（[Ca^2+] i）的瞬时升高（Ca^2+-瞬时升高）是卵激活和进一步发育的先决条件。我们的研究表明，在诱导Ca^2+瞬变的信号通路中，ryanodine系统通过cGMP和cADPR比IP_3系统更上游。在一些细胞中，NO被证明通过激活可溶性鸟苷酸环化酶来刺激cGMP的产生。Kuo等人（2000）提出了一种假设，即NO对于Ca^2+-瞬变的启动是必要的和充分的。测定了受精过程中卵细胞内[Ca^<2+>]_i、胞内NO浓度（[NO]_i）、卵内cGMP和IP_3含量的变化。（1）抑制NO合成酶可使Ca^<2+>瞬变潜伏期稍延长，cGMP和IP_3含量的增加明显延迟，但不能消除Ca^<2+>瞬变。（2）抑制NO敏感的鸟苷酸环化酶可显著延长Ca^<2+>-瞬变的潜伏期，消除cGMP的初始升高，延迟IP_3的升高，降低IP_3的峰值。（3）NO清除剂、氧合血红蛋白对Ca^<2+>-瞬变无明显影响。4)同时测定[Ca^<2+>] i和[NO] i不能检测到受精电位开始前后或Ca^<2+>瞬变开始前[NO] i的增加。5）阻断Ca^<2+>通道，尤其是精子的Ca ^<2+>通道，同时抑制NO敏感的鸟苷酸环化酶或IP_3受体，可显著延长Ca^<2+>瞬变的潜伏期。因此，我们无法获得支持NO是启动Ca^2+瞬变的必要和充分条件这一假设的证据，但再次证实了cGMP在海胆受精时的Ca^2+瞬变中是重要的。

A transient increase of the intracellular Ca^<2+> concentration ([Ca^<2+>]_i)(Ca^<2+>-transient) at fertilization of sea urchin eggs is prerequisite for the egg activation and further development. We have showed the possibility that the ryanodine system via cGMP and cADPR exists more upstream than the IP_3 system in the signaling pathway inducing Ca^<2+>-transients. In some cells, NO is shown to stimulate the production of cGMP by activating soluble guanylyl cyclase. Kuo et al.(2000) have presented the hypothesis that NO is necessary and sufficient for the initiation of Ca^<2+>-transients. We measured the changes of [Ca^<2+>]_i, the intracellular NO concentration([NO]_i), the egg contents of cGMP and IP_3 during fertilization, and obtained the following results. 1)An inhibition of NO synthase slightly elongated the latent period of Ca^<2+>-transients, evidently delay the increase of cGMP and IP_3 contents, but could not abolish the Ca^<2+>-transient. 2)An inhibition of NO-sensitive guanylyl cyclase significantly elongated the latent period of Ca^<2+>-transients, abolished the initial increase of cGMP, delay the increase of IP_3, and diminished the peak of IP_3. 3)NO scavenger, oxyhemoglobin had little effects on the Ca^<2+>-transient. 4)Simultaneous measurements of [Ca^<2+>]_i and [NO]_i could not detect the increase of [NO]_i around the onset of fertilization potential or before the onset of Ca^<2+>-transient. 5)A block of Ca^<2+> channel, especially that of sperm, accompanied with the inhibition of NO-sensitive guanylyl cyclase or IP_3 receptor dramatically elongated the latent period of Ca^<2+>-transient. Thus, we could not obtained the supporting evidences for the hypothesis that NO is necessary and sufficient for the initiation of Ca^<2+>-transients, but confirmed again that cGMP was important in the Ca^<2+>-transient at fertilization of sea urchins.